Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
multi-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: very concise study summary is available

Data source

Reference
Reference Type:
publication
Title:
Die Verträglichkeit der Benzoesäure in chronischen Fütterungsversuch
Author:
Kieckebusch W, Lang K
Year:
1960
Bibliographic source:
Arzneimittel Forschung 10, 1001-1003

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
4-generation study
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Reference substance name:
Benzoic acid
EC Number:
200-618-2
EC Name:
Benzoic acid
Cas Number:
65-85-0
IUPAC Name:
benzoic acid
Details on test material:
- Name of test material (as cited in study report): Benzoesäure

Test animals

Species:
rat
Strain:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Farbwerke Bayer (Elberfeld)
- Age at study initiation: no data
- Weight at study initiation: 40-50 g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): first 8 weeks "paired feed" technique was applied and after ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: no data
- Further matings after two unsuccessful attempts: yes
- After successful mating each pregnant female was caged (how): no data
- Any other deviations from standard protocol: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
every 6 weeks
Duration of treatment / exposure:
Generation 1 and 2: lifelong; generation 3: 16 weeks; generation 4: until breeding
Frequency of treatment:
in 8 first weeks a "paired feed" technique was applied and after ad libitum
Details on study schedule:
no data
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.5, 1 %
Basis:
nominal in diet
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: in the first 8 weeks body weight was measured weekly and later in four week interval

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
no data
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: total number of offspring, % of survived offspring in litter

GROSS EXAMINATION OF DEAD PUPS:
no data
Postmortem examinations (parental animals):
no data
Postmortem examinations (offspring):
no data
Statistics:
no data
Reproductive indices:
no data
Offspring viability indices:
no data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
no effects

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
no effects

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
no effects

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
no data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
no data

ORGAN WEIGHTS (PARENTAL ANIMALS)
no effects: except in 3rd generation females liver weight increase caused by weight increase during lactation and variation in liter size

GROSS PATHOLOGY (PARENTAL ANIMALS)
no effects

HISTOPATHOLOGY (PARENTAL ANIMALS)
no effects

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 1% of benzoic acid in food, assuming 150 mg (1%) of benzoic acid per day per rat and 300 g bw of rat
Dose descriptor:
NOEL
Effect level:
557 mg/kg bw/day (nominal)
Based on:
other: methyl benzoate
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
no effects

CLINICAL SIGNS (OFFSPRING)
no effects

BODY WEIGHT (OFFSPRING)
no effects

SEXUAL MATURATION (OFFSPRING)
no data

ORGAN WEIGHTS (OFFSPRING)
no effects

GROSS PATHOLOGY (OFFSPRING)
no effects

HISTOPATHOLOGY (OFFSPRING)
no effects

Effect levels (F1)

open allclose all
Dose descriptor:
NOEL
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOEL
Generation:
F1
Effect level:
557 mg/kg bw/day (nominal)
Based on:
other: methyl benzoate
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1. Body weight gain in g (mean ± deviation)

 

Control

0.5% Benzoic acid

1% benzoic acid

 

Male

Female

Male

Female

Male

Female

I Generation

 

 

 

 

 

 

Week 4

70 ± 5.0

67 ± 4.1

69 ± 2.7

64 ± 2.4

75 ± 1.4

61 ± 2.2

Week 8

159 ± 6.6

124 ± 5.2

160 ± 5.0

135 ± 5.4

147 ± 2.8

133 ± 2.4

Week 12

219 ± 6.3

-

221 ± 5.6

-

237 ± 6.0

-

 

 

 

 

 

 

 

II Generation

 

 

 

 

 

 

Week 4

69 ± 2.3

57 ± 3.3

71 ± 2.2

60 ± 6.7

73 ± 3.3

70 ± 2.4

Week 8

167 ± 2.0

122 ± 3.6

161 ± 2.6

131 ± 3.5

158 ± 4.6

123 ± 5.3

Week 12

243 ± 5.5

-

238 ± 3.3

-

231 ± 5.6

-

 

 

 

 

 

 

 

III Generation

 

 

 

 

 

 

Week 4

73 ± 3.0

60 ± 3.5

68 ± 3.8

71 ± 2.4

72 ± 3.5

67 ± 3.7

Week 8

157 ± 4.5

134 ± 4.1

158 ± 5.3

137 ± 3.9

168 ± 2.0

136 ± 4.5

Week 12

240 ± 4.6

-

226 ± 5.8

-

233 ± 3.8

-

 

 

 

 

 

 

 

IV Generation

 

 

 

 

 

 

Week 4

93 ± 1.7

76 ± 4.8

84 ± 2.9

70 ± 3.1

73 ± 5.3

72 ± 3.5

Week 8

189 ± 5.3

137 ± 4.3

168 ± 3.7

143 ± 2.4

159 ± 6.2

134 ± 4.5

Week 12

255 ± 4.6

-

245 ± 4.4

-

240 ± 4.4

-

Table 2. The organ weights of animals of the 3rd generation (% of bodyweight)

 

Control

0.5% Benzoic acid

1% Benzoic acid

 

Male

Female

Male

Female

Male

Female

Bodyweight, g

326 ± 5

262 ± 5

329 ± 5

276 ± 5

316 ± 3

248 ± 6

Brain

0.57 ± 0.01

0.69 ± 0.02

0.58 ± 0.01

0.68 ± 0.01

0.61 ± 0.00

0.75 ± 0.02

Heart

0.34 ± 0.02

0.35 ± 0.01

0.32 ± 0.01

0.39 ± 0.01

0.33 ± 0.01

0.35 ± 0.01

Liver

3.49 ± 0.1

5.32 ± 0.3

3.34 ± 0.1

5.19 ± 0.2

3.49 ± 0.1

5.40 ± 0.2

Spleen

0.21 ± 0.00

0.25 ± 0.01

0.21 ± 0.04

0.23 ± 0.01

0.22 ± 0.01

0.25 ± 0.01

Rein

0.72 ± 0.02

0.8 ± 0.03

0.70 ± 0.04

0.79 ± 0.01

0.74 ± 0.02

0.78 ± 0.02

Testes

0.85 ± 0.03

-

0.83 ± 0.04

-

0.92 ± 0.01

-

Table 3. Reproduction in rats in chronic benzoic acid feeding throughout 4 generations

 

Benzoic acid in food, %

 

0

0.5

1.0

Total number of pregnant females

80

78

80

Sterile females, %

14

10

4

Females with delayed sexual maturity

7.5

17

9

Litter per generation

9.0 ± 0.33

9.5 ± 0.26

9.6 ± 0.29

Total number of offspring

625

688

741

% of survived offspring in litter

74

66

65

Old pairing (2nd generation) the number of pregnant females

37

38

38

Litter per generation

6.8

7.7

7.5

Total number of offspring

173

139

171

% of survived offspring in litter

72

61

73

Applicant's summary and conclusion

Conclusions:
No effects were noted after chronic dietary exposure of rats (parents and offspring) to 1% benzoic acid (equivalent to 500 mg/kg bw/day). The NOEL for benzoic acid can be converted to a NOEL for methyl benzoate of 557 mg/kg bw/day.
Executive summary:

In a life long chronic multi-generation study 2 experimental groups of 20 male and 20 female rats were treated with 0.5 and 1% of benzoic acid in food. The control group received untreated food and consisted also of 20 male and 20 female rats. The first and the second generations were treated lifelong, the 3rd generation for 16 weeks and the 4th generation until breeding. No effects on body weight and food intake were detected in all 4 generations. Liver weight increase in females of the 3rd generation was considered as not treatment related effect but may be caused by weight increase during lactation and due to variation in litter size. Otherwise no effects after histopathological examinations were detected in the treated animals of the 3rd generation. No effects were detected on reproduction and offspring raise in both dosing groups. Causes of death in this chronic study were not investigated. Based on the data of this study, the NOEL is 1% (500 mg/kg bw/day of benzoic acid and 557 mg/kg bw/day of methyl benzoate).

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